Actin filaments constitute one of the main components of the cell cytoskeleton. Actin filaments are polymers which spontaneously self-assemble into cells from actin monomers. They form complex intracellular structures, providing mechanical support for regulating cells' shape. Assembled into bundles in filopodia, or in stress fibres, they play a pivotal role during cell morphogenesis, adhesion and motility. The bundles' emergence has been extensively related to specific actin regulators in vivo. Such dynamic modulation was also highlighted by biochemical reconstitution of actin network assembly, in bulk solution or with biomimetic devices. However, the question of how geometric boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures has not been examined.
Monomers of actin can be isolated and purified from filtrated cellular homogenates. In vitro, these monomers can polymerize in the presence of nucleation proteins and ATP (energy source), forming new actin filaments (F-actin). Unlike intracellular assemblies, the filaments network has no particular structure when formed in vitro. Several methods have been described for recreating an actin network in vitro (Haraszti et al., 2009).
In a first approach, the in vitro polymerization of actin filaments is performed in solution from purified monomers (e.g., US2004/0038323, US2006/0003399). However, this type of reconstitution suffers from spontaneous and random organization of the filaments network, in particular the complete lack of geometric control of the initiating regions.
In a second approach, the actin polymerization is performed with beads coated with nucleating proteins dispersed in a solution (Michelot et al., 2007). However, one of the major limitations of these coated beads is their random relative positions in solution.
In a third approach, the filaments' organization is controlled after their polymerization in a solution.
For instance, they may be immobilized on a pillar network (i.e., the actin filaments are put in a line with the pillar network) (Roos et al., 2003). Arrays of 2-micron-wide gold discs were fabricated on top of 20-micron-high pillars. These dots were used to graft myosin and thereby attach actin filaments. These attached filaments were then elongated using a solution comprising actin monomers. Long filaments growing out of the dots were not oriented and the network was not spontaneously organized in space. But the addition of filamin, an actin associated protein, could force filament bundling and induce the formation of bridges between the dots. This organization could occur only when filaments were anchored on top of micro-pillars, whose length has to exceed the filaments' length. The same array of dots on a flat surface, rather than on top of pillars, only promotes the formation of an ill-defined network even in the presence of filamin.
They may also be sent in a chamber where beads with optical traps are placed (Uhrig et al., 2009).
However, in these two cases, the actin filament network is not self-assembled; it is externally oriented by fluid flow or capturing beads' positions. The final architecture does not rely on biological properties of actin filaments assembly and interactions, so these methods cannot be used to test these properties. In addition, in the filament bundles that are externally oriented, the polarity of individual filament is not controlled. The precise architecture of these networks is therefore not highly controlled.
In a fourth approach, the filaments' organization is controlled on a solid support by introducing on a surface an actin nucleation site and an actin capture site (US20050106629). With these two anchorage points, the filaments' organization may be controlled. However, the structure is not self-assembled but externally arranged by the controlled location of nucleation and capture sites. In particular, the nucleation sites are arranged on the support so that the actin filaments from one nucleation site do not interact with actin filaments from another nucleation site. In addition, filament growth out of the nucleation site is not oriented and has to be externally driven by beads, or manipulated by a magnetic or optical trap.
None of these approaches provide self-assembling of an ordered actin filament network with reproducible and controlled geometry.